A Cyclic GMP–Dependent Calcium-Activated Chloride Current

Home , Bay K8644, Calcium channel opener, Pinacidil

The Journal of General Physiology

(45) 86129065;email:[email protected] Universitetsparken, Building160, DK-8000AarhusC,Denmark.Fax: Salt ResearchCenter,Department ofPhysiologyUniversityAarhus Address correspondencetoVladimir V.Matchkov,TheWaterand cium release.ThiscGMP-dependentCa dent oncyclicGMPandactivatedbyintracellularcal- deﬁne amembranecurrentthatwascriticallydepen- tosolic oscillator(Pengetal.,2001).We wereableto membrane potentialvariationsbeingdrivenbythecy- tracellular oscillatorsinthesmooth-musclecells,with tosynchronizein- membrane potentialvariationsserve enced bytheendothelium.We havesuggestedthat smooth-muscle levelsofcGMPandthereforeinﬂu- small arterieswasalsocharacteristicallydependenton and membranepotentialvariations.Vasomotion inthe from intracellularstores)(BerridgeandGalione,1988) play betweenacytosolicoscillator(calciumrelease tone ofvessels—vasomotion—originatedfrominter- that theregularorirregularoscillationindiameter 1993; GustafssonandNilsson,1994),itwassuggested In previousworkonsmallarteries(Gustafssonetal., Smooth-muscle CellsfromRatMesentericResistanceArteries A CyclicGMP–dependentCalcium-activatedChlorideCurrentin INTRODUCTION

Vladimir V.Matchkov,ChristianAalkjaer, nist Rp-8-Br-PET-cGMP orwithapeptideinhibitorofPKG,thenonhydrolysableATP analogueAMP-PNP. vated bytheconstitutivelyactivesubunitofPKG.CurrentactivationwasblockedproteinkinaseGantago- smooth-muscle contraction.Herewedemonstratethecharacteristicsofthiscurrent.Usingconventionalpatch- vated inwardcurrentinvascularsmooth-musclecells,andsuggestedthistobeofimportancesynchronizing key words: F Under biionicconditions,theanionpermeabilitysequenceofchannelwasSCN activated chloridecurrentwithanabsoluterequirementforcyclicGMP(EC omycin, orbyhighcalciumconcentration(900nM)inthepipettesolution.Thecurrentwasfoundtobeacalcium- arteriesbyelevationofintracellularcalciumwitheither10mMcaffeine,1 resistance clamp technique,whole-cellcurrentswereevokedinfreshlyisolatedsmooth-musclecellsfromratmesenteric abstract The Water andSalt ResearchCenterandDepartmentofPhysiology, UniversityofAarhus, DK-8000Aarhus, Denmark other calcium-activatedchloridecurrents. chloride current,whichisactivatedbyintracellularcalciumreleaseandhascharacteristicsdistinctfrom that smooth-musclecellsfromratmesentericresistancearterieshaveanovelcGMP-dependentcalcium-activated current similartothelattercouldbeidentified alsointhemesentericarterysmooth-musclecells.We conclude has previouslybeenreportedforthiscurrent.Underconditionsofhighcalciuminthepatch-pipettesolution,a sensitivity toinhibitionbyniflumic acid,wasunaffectedbyzincions,andshowedoutwardcurrentrectification as the calcium-activatedchloridecurrentinpulmonary myocytes,whichwascGMP-independent,exhibitedahigh DIDS, andIAA-94.Thepropertiesofthiscurrentinmesentericartery smooth-musclecellsdifferedfromthoseof was unaffectedbycobaltandhadalowsensitivitytoinhibitionthechloridechannelblockersniflumic acid, current hadnovoltageortimedependence.Itwasinhibitedbynickelandzincionsinthemicromolarrange,but

aspartate,buttheconductancesequencewasI

We havepreviouslydemonstratedthepresenceofacyclicGMP(cGMP)-dependentcalcium-acti-

chloride channels•calciumsignalingoscillationscyclicnucleotides

121

oue13Fbur 04121–134 February 2004 Volume 123 http://www.jgp.org/cgi/doi/10.1085/jgp.200308972 J. Gen.Physiol.

2 ©TheRockefellerUniversityPress -activated (I to combinepropertiescharacteristic fordifferentfami- 1999; Greenwoodetal.,2001). Thenewchannelseems tail previously(Yuan, 1997;GreenwoodandLarge, terial myocytes,whereithas beencharacterizedinde- therefore alsocharacterizedI fers inmanyoftheseaspects.Forcomparisonwehave cells frommesentericsmallarteriesdescribedheredif- cGMP-dependent chloridecurrentinsmooth-muscle masa andNorth,1996).However, thepropertiesof and speciﬁcinteractionswithmultivalentcations(Toki- permeability sequenceforhalides(Fringsetal.,2000), niﬂumic acid(LargeandWang, 1996),acharacteristic dence, sensitivitytopharmacologicalblockerssuchas current haspropertiessuchastimeandvoltagedepen- 1996; Fringsetal.,2000).Thepreviouslycharacterized from previouslydescribedI rent wasshowntobeaCa istics ofthisCa potential andintracellularcalciumrelease. ticipate inthereciprocalinteractionofmembrane current seemedtohaveallpropertiesrequiredpar- and

Cl(Ca) In thepresentworkwehaveexaminedcharacter- Br HolgerNilsson ), ithasseveralcharacteristicsthataredistinct

• 2 0022-1295 acetate -activated current.Althoughthiscur- 50 6.4 /2004/02/121/14 $8.00

M). Thecurrentcouldbeacti- F Br

Cl(Ca) M BAY K8644,0.4 Cl(Ca) 2 aspartate

-activated Cl (LargeandWang, from pulmonary ar- frompulmonary I

SCN acetate - current M ion- . The

lies of Cl channels. Brief preliminary reports of part of Currents were recorded at a holding potential of 60 mV. the present results have been given elsewhere (Match- Current-voltage characteristics were obtained either from ramp kov et al., 2001, 2003). protocols or from a voltage-step protocol. Voltage ramps were performed from 60 to 60 mV with a duration of 100 ms, un- less specified otherwise. In the voltage-step protocol currents MATERIALS AND METHODS were evoked by pipette solutions containing calcium buffered at 900 nM (Fixed-Ca solution, see below). This technique has been Cell Isolation used to activate ICl(Ca) in studies on smooth muscle (Greenwood et al., 2001; Britton et al., 2002) and directly activates the chlo- All experiments were conducted in accordance with the Danish ride current at a clamped concentration of calcium. The current- national guidelines for animal research. Male Wistar rats (12–18- voltage relationship of the activated current (after subtraction of wk-old) were killed with CO2. The mesentery and in some cases baseline current) was determined by stepping the potentials (20- the pulmonary artery was removed from the rats and placed into mV steps) between 90 and 90 mV, maintaining the voltage at an ice-cold physiological salt solution. Branches of small mesen- each step for 2 s. Current amplitudes were averaged over the last teric arteries were dissected out and cleaned from connective tis- 0.1 s before the end of the test pulse. sue. All arteries were opened longitudinally, and endothelium To obtain the relative permeabilities for anions under biionic and remaining blood were removed by gently rubbing the lumi- conditions the current was activated by application of ionomycin nal side of the arteries. in symmetrical chloride conditions. Current-voltage relationships Subsequently, in most experiments papain digestion (Schu- were obtained by 2-s voltage steps covering the range 70 to 90 bert et al., 2001) was used to isolate smooth-muscle cells. The ar- mV in 20-mV increments from a holding potential of 90 mV. teries were stored overnight at 4 C in a solution for enzymatic di- The protocol was performed initially in symmetric, chloride-con- gestion of the arteries (for composition see below). Before exper- taining solution, and then repeated during extracellular superfu- iments, the microtube with the vessels was incubated for 5–10 sion with the substitute anion. Finally, chloride-containing solu- min at 37 C, after which the vessels were transferred to a low- tion was reapplied and a control measurement was made. Cur- calcium solution. Single cells were released by trituration with rent values were taken from the last 0.1 s of the 2-s voltage step. a polyethylene pipette into the experimental solution. This Ionomycin was present throughout. Results were discarded from method consistently produces a high yield of relaxed smooth- analysis if the initial and final measurements in the presence of muscle cells, which respond reversibly to vasoconstrictors and re- chloride differed in characteristics. main viable for at least 3 h (Clapp and Gurney, 1991). In experiments where BAY K8644 was used to activate calcium Drug Application channels, cells were instead isolated by the collagenase-elastase method of Petkov (Petkov et al., 2001). This method produces Drugs were either applied to the bath or superfused locally over smooth-muscle cells that express a high density of voltage-gated the patched cell (except experiments in biionic conditions, calcium currents. where drugs were applied in the solutions for flow exchange with the rate 5 ml/min; see above). For local superfusion, a specially Patch-clamp Recording designed electric manipulator (Danish Myo Technology) was used to position the application pipettes immediately before ap- All experiments were made at room temperature (22–24 C). plication and to move them out of the solution immediately af- Patch pipettes were prepared from borosilicate glass (PG15OT- terwards. In this way seepage of drugs onto the cells before or af- 7.5; Harvard Apparatus), pulled on a P-97 puller (Sutter Instru- ter application was eliminated. ment Co.) and fire polished to achieve tip resistances in the The standard protocol consisted of breaking the membrane range of 2–5 M . and waiting 5 min for equilibration. Then a control recording Recordings were made with an Axopatch 200B amplifier (Axon was made, followed by a 10 min wash out. Finally, the drug was su- Instruments, Inc.) in whole-cell configuration. Data were sampled perfused for 1 min followed by another current recording in the at a rate of 2 kHz and filtered at 1 kHz. Data acquisition and analy- continued presence of the drug. sis were done with the software package Clampex 7 for Windows (Axon Instruments, Inc.). Only cells with essentially no leak current Solutions (seal resistance 2 G) and a low access resistance (5–10 M) were used, and the stability of these parameters was tested regularly The solution used for enzymatic digestion of arteries contained during the course of the experiment. Series resistance and capaci- (in mM): NaCl 110, KCl 5, MgCl2 2, KH2PO4 0.5, NaH2PO4 0.5, tive current were routinely compensated. Junction potentials were NaHCO3 10, CaCl2 0.16, EDTA 0.49, Na-HEPES 10, glucose 10, estimated using the junction potential calculator of pClamp 7 taurine 10 at pH 7.0, as well as 1.5 mg ml1 papain, 1.6 mg ml1 (Axon Instruments, Inc.) and data were corrected for these. albumin, and 0.4 mg ml1 dl-dithiothreitol. The collagenase/

TABLE I Bath Solutions Solution Na K Cs Ca2 Mg2 Ba2 Cl Aspartic acid ChTX HEPES EGTA pH pH adjustment

mM mM mM mM mM mM mM mM mM mM mM mM B1 control 145 6 0.1 1 1 145 104 10 7.4 NaOH B2 control Cs 140 0.1 1 142 104 10 7.4 CsOH B3 low Ca 145 6 0.9 1 1 147 104 10 1 7.4 NaOH B4 low Ca-Cs 140 0.9 1 142 104 10 1 7.4 CsOH B5 Ca free 145 6 1 1 145 104 10 1 7.4 NaOH B6 low Cl 140 0.1 1 67 75 104 10 7.4 CsOH

122 A cGMP-dependent Ca2-activated Cl Channel

TABLE II Pipette Solutions Used Solution Na K Cs Ca2 Mg2 Cl MgATP BAPTA HEPES EGTA cGMP Aspartic acid pH pH adjustment

mM mM mM mM mM mM mM mM mM mM mM mM P1 control 10 132 0.01 1 134 0.1 0.1 10 102 7.35 KOH P2 control Cs 140 0.01 140 0.1 0.1 10 7.35 CsOH P3 high BAPTA 10 112 1 1.44 117 0.1 10 10 102 7.35 KOH P4 high EGTA 10 112 1 1.44 117 0.1 10 11 102 7.35 KOH P5 fixed Ca 140 5 140 0.1 10 6 7.35 CsOH P6 low Cl 140 5 65 0.1 10 6 75 7.35 CsOH elastase enzyme solution contained (in mM): NaCl 110, KCl 5.6, different isolations. Unpaired Student’s t test was used for single MgCl2 1.2, Na-HEPES 10, glucose 20, taurine 20, Na-pyruvate 5 at comparisons, and one-way analysis of variance test with Bonfer- pH 7.4, as well as 1 mg ml1 collagenase type 1 (Worthington Bio- roni’s post-test for multiple comparisons (GraphPad Prism v. chemical Corporation), 4.85 g ml1, elastase type I (Sigma-Aldrich), 2.01; GraphPad Software). Nonlinear regression to the Hill equa- 1 mg ml1 soybean trypsin inhibitor, 1 mg ml1 albumin. tion was used for the analysis of concentration-effect curves. Lin- The active subunit of protein kinase G (PKG) was obtained by ear regression was used to compare estimated and experimental trypsinization of PKG as described by White et al. (1993). In changes in ECl, and in experiments for determination of the rela- brief, 10 g PKG I was dissolved in 300 l water. Trypsin (1 g) tive conductance of halides. was added and incubated for 3 min at 30C, after which 10 g Relative permeability was determined by measuring the shift in soybean trypsin inhibitor was added. This solution was added in a reversal potential (Erev) upon changing the solution on one side ratio of 1:30 to the pipette solution. of the membrane from one containing chloride ions (Cl) to an- Compositions of solutions used in the patch-clamp experi- other with the substitute anion (X). The permeability ratio was ments are given for bath solutions in Table I, and for pipette so- estimated using the Goldman-Hodgkin-Katz equation: PX/PCl lutions in Table II. Free calcium concentration was estimated by exp( ErevF/RT), where Erev is the difference between the re- the WebMaxC v2.22 buffer program (Chris Patton, Stanford Uni- versal potential with the test anion X and that observed with versity, CA). For the “low-calcium” (B3; Table I) and “low-cal- Cl, F is Faraday’s constant, R is the gas constant, and T is tem- cium-Cs” (B4) solutions in the presence of 1 mM BaCl2 free cal- perature. cium was estimated to be 19 M. To determine the relative permeability to halides the CsCl in the extracellular solution was RESULTS substituted with the corresponding cesium salt on an equimolar basis in 4–6 experiments for each ion species tested. In experi- A cGMP-sensitive Calcium-activated Inward Current in ments under biionic conditions 3 M KCl salt-agar bridge was Smooth-muscle Cells placed between the Ag/AgCl pellet and the bath solution. Drugs and Chemicals We have previously reported the presence of a calcium- activated inward current that required cyclic GMP for Adenosine 3,5-cyclic monophosphate (cAMP), adenosine 5- activation in rat mesenteric arterial smooth-muscle ( , -imido)triphosphate tetralithium salt hydrate (AMP-PNP), cells (Peng et al., 2001). Those experiments were ATP--S, albumin, 4-aminopyridine (4-AP), 1,2-bis(2-aminophe- noxy)ethane-N,N,N,N-tetraacetic acid (BAPTA), cesium acetate made using the ampthotericin permeabilized-patch tech- (Cs-acetate), cesium bromide (CsBr), cesium chloride (CsCl), cesium nique. In the present set of experiments, we were able fluoride (CsF), cesium iodide (CsI), guanosine 3,5-cyclic monophos- to identify a similar current in conventional, ruptured- phate (cGMP), caffeine, 4,4-diisothiocyanatstilbene-2,2-disulphonic patch whole-cell recordings. In the presence of 10 M acid (DIDS), dl-dithiothreitol, ethylenediaminetetraacetic acid cGMP in the intracellular solution (solutions B1:P1, as (EDTA), ethylene glycol-O,O-bis(2 aminoethyl)-N,N,N,N-tetraacetic acid (EGTA), hydroxyethylpiperazine-N-2-ethanesulphonic defined in Tables I and II), application of 10 mM caf- acid (HEPES), magnesium adenosine-5-triphosphate (MgATP), feine evoked a transient current in 90% of cells, as nifedipine, niflumic acid, N-methyl-D-glucamine (NMDG), pa- shown in Fig. 1 A. The density of this whole-cell current pain, sodium thiocyanate (NaSCN), tetraethylammonium chlo- at 60 mV holding potential was 7.58 0.35 A F1 ride (TEA), indanyloxyacetic acid (R( )-IAA-94), trypsin, as well (n 57) in cells with an average capacitance of 16.2 as the salts for all solutions were obtained from Sigma-Aldrich. Charybdotoxin (ChTX) and iberiotoxin (IbTX) was purchased 0.47 pF. The time-course of this current was similar to from Latoxan. Mibefradil was obtained from Hoffman-LaRoche; the time-course of the calcium elevation measured by –phenyl-1,N2-etheno-8-bromoguanosine-3-5-cyclic monophos- Fura-2 in response to caffeine (unpublished data). phorothioate, Rp-isomer (Rp-8-Br-PET-cGMP) from BioLog Life Chelating the intracellular calcium with either 10 mM science Institute; apamin from Alomone Labs; protein kinase BAPTA (n 8) or 11 mM EGTA (n 6) (solutions B1: G I and cell-permeable protein kinase G I inhibitor from Calbio- chem; soybean trypsin inhibitor from Fluka. P3 and B1:P4, respectively) eliminated this current. This observation was consistent with the effect of 10 Statistics M ryanodine seen in previous experiments (Peng et All data are given as means SEM. Only one experimental re- al., 2001) and showed that the evoked current was sec- cording was taken from each cell, thus n is the number of cells. ondary to the calcium elevation by caffeine. In the ab- Statistical analysis was performed using cells from at least three sence of cGMP, no current or a small inward current 123 Matchkov et al. was observed in response to caffeine application at 60 mV holding potential: current density without cGMP was 0.12 0.14 A F1 (n 30) in cells with an average capacitance of 18.9 4.1 pF. A similar current could be evoked by increasing calcium influx in the presence of ryanodine, by applying either ionomycin or the calcium channel opener, BAY-K 8644. Both treatments caused sustained elevations in intracellular calcium measured by Fura-2 (unpublished data). Bath application of 0.4 M ionomycin (solutions B4:P2 with 10 M ryanodine added to the pipette solution) caused only small changes in current, but subsequent application of membrane-permeable 8Br-cGMP rapidly activated a sustained inward current (Fig. 1 B) with an average amplitude of 10.3 1.12 A F1 (n 14) at a holding potential of 60 mV in cells with an average capacitance of 16.0 1.0 pF. The sequence of events were similar after application of first BAY-K 8644 and then 8Br-cGMP (Fig. 1 C); average current density here was 6.48 0.41 A F1 (n 5) (60 mV holding potential; cell capacitance 23.0 2.85 pF). Run-down of the current in the presence of cGMP was not pronounced (unpublished data), suggesting that it did not critically depend on components in the cytoplasm that could be washed out in the whole-cell experiment.

Ion Selectivity The inward current is unlikely to be a potassium current, since the potassium equilibrium potential (78.5 mV) is negative to the holding potential (60 mV). Also, the cGMP-sensitive inward current was insensitive to various K-channel antagonists: neither 100 nM charybdotoxin (n 34) nor 100 nM iberiotoxin (n 13), 1 M apamin (n 5), 10 mM 4-aminopyridine (n 5), or 10 mM tet- Figure 1. Original recordings of whole-cell cGMP-dependent in- raethyl ammonium (n 5) reduced the [Ca2] -acti- ward current in response to a 10-mM caffeine application (A), to i 0.4 M of ionomycin (B), and to 1 M of the voltage-gated cal- vated inward current. In 6 of 10 experiments performed cium channel agonist BAY K8644 (C). The holding potential in all in the absence of potassium-channel blockers, a tran- experiments was 60 mV. Solutions were: A, B1:P1; B and C, B4: sient outward current was seen superimposed on the in- P2 with 10 M ryanodine added to the pipette solution. ward current: this outward current was most likely due to activation of a KCa channel, since it was never observed in the presence of 100 nM charybdotoxin (ChTX). Calcium ions could also potentially carry inward It is possible that Na ions carried the inward current current (ECa 89 mV for B1:P1). However, neither 1 (ENa 68 mV for B1:P1). However, neither partial nor M nifedipine (n 5) nor 10 M mibefradil (n 5) complete replacement of extracellular sodium affected inhibited the inward current. Furthermore, superfus- the amplitude of the current, indicating that sodium is ing the cell for 1 min with a Ca2-free solution before not involved: the inward current amplitude was 8.66 caffeine application did not affect the current: the in- 0.74 A F1 (n 6; cell capacitance 15.8 0.65 pF) in the ward current amplitude was 8.12 2.12 A F1 (n 5; presence of 145 mM Na (solutions B1:P1), 8.93 0.62 solutions B1:P1) in the control conditions and 8.79 A F1 (n 4; cell capacitance 16.9 2.59 pF) in the 2.07 A F1 (n 5; cell capacitance 12.8 0.46 pF) presence of 10 mM Na (the bath solution B1 was modi- after superfusion with Ca2-free solution (solutions fied by substitution of 135 mM Na with NMDG; the pi- B5:P1). This finding not only demonstrates that the pette solution was P1), and 7.15 0.48 A F1 (n 5; cell charge carrier is not calcium ions, but it also shows capacitance 14.8 1.71 pF) in the absence of Na (the that the calcium that activates the current during caf- bath solution B1 was modified by complete substitution feine stimulation is derived from an intracellular of Na with NMDG; the pipette solution P1). pool, and that the current under these conditions is

124 A cGMP-dependent Ca2-activated Cl Channel Figure 2. The relation between measured reversal potential of the current and calculated equilibrium potential for chloride (ECl). The dashed line is the identity line, expected for an ideal chloride channel. Solutions were B2:P6, B2:P5, and B6:P5 from left to right points, respectively. 10 M cGMP was added to all pipette solutions. Vertical bars indicate SEM. not activated secondarily to calcium influx through, e.g., store-operated calcium channels. A further candidate as charge carrier for an inward current is chloride. An involvement of chloride was tested in CsCl solutions by partially replacing chloride by aspartate in either the intracellular or extracellular solution. The replacement was such that it would move the equilibrium potential 20 mV in either direction. This caused corresponding shifts in the reversal potential of the current (Fig. 2). The current thus was determined to be carried by chloride ions. Effects of Chloride Channel Blockers Since the current appeared to be a chloride current, we examined the effect of the classical chloride channel blockers niflumic acid, IAA-94, and DIDS on the current. Fig. 3 shows the caffeine-evoked inward current before and after a 1-min preincubation with the blockers. While the current could be inhibited by relatively high concentrations of these blockers, it was less sensitive than expected for a typical calcium-activated chloride current. Figure 3. Concentration-dependent effects of the Cl channel antagonists niflumic acid (A), IAA-94 (B), and DIDS (C) on the in- Effect of Divalent Metal Ions ward current response to caffeine. Solutions were B1:P1. Vertical bars indicate SEM. Since some chloride currents have been reported to be sensitive to divalent cations, we tested Ni2, Zn2, and Co2 on the caffeine-activated current. mM caffeine application, by incubation with 0.4 M A 1-min incubation with 100 M Zn2 inhibited the ionomycin, or by an application of 1 M BAY K 8644 calcium-activated inward current evoked either by a 10- (Fig. 4, A and B). Even in a concentration of 1 M,

125 Matchkov et al. Figure 4. Effect of divalent cations on the cGMP-dependent Ca2-activated Cl current. A shows the mean effect of ZnCl2 on the cGMP-sensitive ICl(Ca) evoked by caffeine, ionomycin, and BAY K 8644. Solutions were B1:P1, B4:P2, and B4: P2, from left to right. 10 M cGMP was added to all pipette solutions. Vertical bars indicate SEM. Original recording in B shows two control responses to 10 mM caffeine (black bars) and one response in the presence of Zn2. Solutions were B1:P1. The breaks in the recordings correspond to 10-min wash out periods. Trace in C shows original recordings of the currents evoked by 0.4 M ionomycin application in solutions B4:P2 with 10 M cGMP added to the pipette solution. The concentration of ZnCl2 in the bath was cumulatively increased. D shows concentration-response relation for Zn2 constructed from experiments as shown in C. Vertical bars indicate SEM. E shows concentration-response relation for Ni2 on the inward current response to caffeine. Vertical bars indicate SEM. Solutions were B1:P1.

2 2 Zn strongly reduced the current (tested only with caf- had an EC50 for Zi of 2.2 M with a Hill coefficient of feine; Fig. 4 A). The effect of Zn2 was specific for the 1.0. (Fig. 4, C and D). The high Zn2 sensitivity was used chloride current, since KCa channel currents could still to identify this current in later experiments. be activated by caffeine application if the experimental Also Ni2 inhibited the calcium-activated inward cur- solutions did not contain K channel blockers (bath so- rent, although less potently then Zn2. As shown in Fig. lution was B1 without BaCl2 and ChTX; pipette solution 4 E, the concentration-effect curve had an EC50 for was P1): at 60 mV, outward current density was 159 Ni2 of 27.5 M with a Hill coefficient of 1.5. In con- 38 A F1 (n 4; cell capacitance 17.9 3.85 pF) in the trast, Co2 had little effect on the current at concentra- absence of Zn2 and 177 40 A F1 (n 4, NS; cell cations up to 100 M. The current density was 6.28 pacitance 17.8 2.69 pF) in the presence of 100 M 1.10 A F1 under control conditions, and after 1 min in Zn2, and this was reduced to 0.6 0.9 A F1 (n 4; the presence of 100 M Co2 it was 5.70 1.07 A F1 cell capacitance 15.3 1.45 pF) by further addition of (n 4, NS; cell capacitance 17.8 2.23 pF) when the 100 nM ChTX. Thus, the effect of Zn2 is an inhibition membrane potential was held at 60 mV. of the inward current evoked by calcium release, and Anion Selectivity of the Current not an inhibition of the calcium release itself. The concentration-effect curve for the effect of Zn2 We obtained the relative permeabilities for different on the inward current evoked by ionomycin in solutions anions under biionic conditions (see materials and B4:P2 (with 10 M cGMP added to the pipette solution) methods) based on symmetrical CsCl solution with ex-

126 A cGMP-dependent Ca2-activated Cl Channel Figure 5. Typical current-voltage recordings under biionic conditions, representative of 4–6 experiments of each kind. ICl(Ca) was stimulated by ionomycin in solutions B4:P2 with 10 M cGMP added to the pipette solution. Chloride was substituted with ﬂuoride (A), bromide (B), iodide (C), thiocyanate (D), acetate (E), and l-aspartate (F).

tracellular ionomycin. Typical current-voltage record- P5) often caused the appearance of a current even in ings are shown in Fig. 5, A–F. As is evident from the cur- the absence of cGMP. This was in contrast to experi- rent-voltage curves, the chloride current did not show ments with either caffeine, ionomycin, or BAY-K 8644, any voltage dependence or rectification. The relative where such a current was never observed. This was ana- permeability (PX/PCl) was calculated from the shift in lyzed further in the experiment in Fig. 6 A. Here, cal- Erev using the Goldman-Hodgkin-Katz equation and the cium was introduced from the pipette solution, and relative conductance (GX/GCl) was measured for out- voltage steps showed the presence of a slowly activating ward currents. The selectivity of the channel estimated current, with a reversal potential close to ECl. This cur- from PX/PCl followed the sequence: SCN Br I rent was sensitive to inhibition by niflumic acid. Subse- Cl acetate F aspartate (2.85:1.39:1.25:1:0.87: quent addition of 8Br-cGMP caused the appearance of 0.79:0.06). However, the relative conductances exhib- another current without time dependence that was sen- ited a different sequence: I Br Cl acetate sitive to inhibition by Zn2. This protocol revealed the F aspartate SCN (1.41:1.17:1:0.85:0.71:0.26:0.23). presence of a conventional calcium-activated Cl cur- Based on these results we estimated the relative affinity rent in the same cells that expressed the cGMP-depen- of the anion for a binding site in the channel (Qu and dent current. Hartzell, 2000) as the ratio of the relative permeabili- Inspection of the tail currents in Fig. 6 A showed the ties to the relative conductances; this sequence was: expected tail current of the conventional calcium-acti- SCN Br F Cl acetate I aspartate. vated current (first panel), but the possible increase in tail current after application of 8Br-cGMP is not ex- Current Activation pected for a current lacking time and voltage depen- We noticed that elevating calcium via the pipette solu- dence. This observation prompted us to examine the tion (900 nM free calcium in Cs-containing solution, tail current more closely in experiments using ionomy-

127 Matchkov et al. Figure 6. Activation characteristics of Ca2-activated Cl currents in mesenteric artery smooth-muscle cells. (A) A cGMP-independent current was activated by depolarization in the presence of 900 nM Ca2 in the pipette solution (solutions were B2:P5). This current showed slow activation in response to a depo- larizing step (left) and was inhibited by 100 M niflumic acid (second panel). In the presence of niflumic acid, addition of 300 M 8Br-cGMP revealed the presence of another inward current (third panel), which was inhibited by 10 M Zn2 (right). (B) Superfusion with 0.4 M ionomycin (solutions B4:P2 with 10 M cGMP added to the pipette solution) caused appearance of only the cGMP- sensitive current, which activated immediately and deacti- vated without a tail current. cin to elevate intracellular calcium (since this proce- The possible involvement of a kinase in the activation dure only activated the cGMP-dependent current). As of the current was investigated by replacing the ATP shown in Fig. 6 B, under these conditions an instantly normally present in the intracellular solution by the activating current was seen that lacked tail current. The nonhydrolyzable AMP-PNP. As shown in Fig. 7 E, in the cGMP-dependent current thus seems to be instant-on, presence of 3 mM AMP-PNP 20% of the current instant-off; the reason for the apparent increase in tail could be evoked by caffeine. The lack of effect of the current in Fig. 6 A is unknown. competitive inhibitor AMP-PNP in lower concentration (0.1 M) may indicate sufficient production of ATP within the cell to sustain current activation. Cyclic GMP Dependence Further support for the involvement of a kinase in ac- As mentioned above, hardly any inward current could tivating the current was obtained by studying the effects be evoked by caffeine in the absence of cGMP. Increas- of ATP--S, which can replace ATP as a kinase sub- ing concentrations of cGMP increased the amplitude of strate. The thiol group of ATP--S is ineffective as a sub- the current (Fig. 7 A; solutions B1:P1 with the concen- strate for phosphatases (Yount, 1975), leading to what trations of cGMP shown in Fig. 7 A added to the pipette functionally appears as irreversible phosphorylation. solution). The concentration-effect curve was quite Including ATP--S in the pipette solution caused the steep, with a Hill coefficient of 3.3 and an EC50 of appearance of an inward current over the first few min- 6.4 M. Based on this concentration-effect relation, utes after breaking into the cell (Fig. 7 F). When this we routinely used 10 M internal cGMP to activate the current had stabilized, addition of 8Br-cGMP had no current near-maximally. further effect (not depicted). The current was inhib- The effects of cGMP were further investigated phar- ited by 100 M Zn2. Thus, ATP--S activated the macologically. The current evoked in the presence of 10 cGMP-dependent current as would be expected if a ki- M cGMP was antagonized by the competitive cGMP an- nase was involved. tagonist, Rp-8-Br-PET-cGMP (Fig. 7 B), as well as by the We also examined whether PKG was capable of acti- membrane-permeable PKG inhibitor developed by Dost- vating the current, using the active subunit of PKG iso- mann et al. (2000) (Fig. 7 C). Current activation was se- lated as described in materials and methods. When lective for cGMP: even in 10-fold higher concentrations this was added to a pipette solution without cGMP, acti- than required for cGMP, the activation by cAMP was only vation of a Zn2-sensitive current was observed after 10% of maximal, and the cAMP effect was inhibited by breaking into the cell (Fig. 7 F). Activation was not seen the cGMP antagonist, Rp-8-Br-PET-cGMP (Fig. 7 D). if ATP was replaced by 3 mM AMP-PNP in the pipette

128 A cGMP-dependent Ca2-activated Cl Channel Figure 7. Characterization of the effects of cGMP. (A) Concentration-effect relation for cGMP on the caffeine-activated inward current. EC50 is 6.4 M, the Hill coefficient is 3.3. Solu- tions were B1:P1 with addition of different cGMP concentrations to the pipette solution. (B) Con- centration-effect relation for inhibition of the inward current induced by caffeine by the competitive cGMP antagonist, Rp-8-Br-PET-cGMP. Note near-complete inhibition of current at high antagonist concentration. The EC50 corresponds to 37 M, and the Hill coefficient is 2.3. Solutions were B1:P1. (C) Inhibition of the caffeine-induced inward current by a membrane-permeable peptide inhibitor of PKG (Dostmann et al., 2000). Solu- tions were B2:P2 with 10 M cGMP added to the pipette solution. (D) Comparison of effects of cGMP and cAMP on the caffeine-induced inward current. Even the 10-fold higher concentration of cAMP activated only a fraction of the current, and this was inhibited by the cGMP antagonist. Solu- tions were B1:P1 with either 10 M cGMP or 100 M cAMP added to the pipette solution. (E) Inhi- bition of the caffeine-induced inward current by the nonhydrolyzable ATP analogue, AMP-PNP. The bath solution was B1. The pipette solution was P1 (containing 10 M cGMP), in which ATP was replaced by the indicated concentration of AMP-PNP. (F) Activation of the membrane current by irreversible phosphorylation with ATP--S and by intracellular application of the active subunit of PKG. Both agents cause the activation of the zinc-sensitive current. Solutions were B2:P5. In A–F the number of experiments (n) is indicated near each point or bar. Vertical bars indicate SEM. solution (unpublished data). Adding 8Br-cGMP in the artery myocytes (current density 6.93 1.53 A F1, n presence of active PKG did not enhance the current 5; capacitance 15.8 1.6 pF). Also in contrast to the further (unpublished data). This demonstrates that the cGMP-dependent current in the mesenteric artery current can be activated by PKG. smooth-muscle cells, the current in the pulmonary artery myocytes was inhibited (n 5) by 100 M niflumic Calcium-activated Chloride Current in Pulmonary acid (Fig. 8). Artery Myocytes The current-voltage relations were obtained for the To confirm that the characteristics of the cGMP-depen- current evoked by caffeine using a ramp protocol (Fig. dent current are not merely a reflection of our experi- 8 B). The current exhibited outward rectification as mental conditions, we also recorded the previously previously described for ICl(Ca) in pulmonary arteries (Large and Wang, 1996; Greenwood et al., 2001). well-characterized ICl(Ca) from pulmonary arteries (Sal- ter et al., 1995; Greenwood and Large, 1999; Green- wood et al., 2001). This current could be evoked by DISCUSSION caffeine in the absence of cGMP (Fig. 8 A, solution B1:P1 without cGMP in the pipette solution). The den- In the present work we have characterized a cGMP- sity of the whole-cell current at 60 mV in the control dependent, Ca2-activated inward current that we have condition was 6.16 0.74 A F1 (n 14; capacitance previously demonstrated (Peng et al., 2001) in vascular 18.5 1.5 pF) and was not enhanced by 300 M 8Br- smooth-muscle cells from rat mesenteric small arteries. cGMP (5.91 1.06 A F1, n 5; capacitance 19.8 1.8 The most prominent channel activated by calcium re- pF). Zn2 at a concentration of 100 M, which elimi- lease in vascular smooth-muscle is the large-conduc- nates the cGMP-dependent ICl(Ca) of mesenteric artery tance calcium-activated potassium channel (ZhuGe et myocytes, did not change the current in the pulmonary al., 1998; Smani et al., 2001). Such a current was also

129 Matchkov et al. Figure 8. Ca2-activated Cl currents recorded in smooth-muscle cells from pulmonary arteries. Original recordings in A show stimulations by 10 mM caffeine (as indicated with black bars) under control conditions (solutions were B1:P1 without cGMP in the pipette solution) and in the presence of 8Br-cGMP, ZnCl2, and niflumic acid as indicated. Breaks in the records correspond to 10 min wash periods. B shows the mean current densities of the Ca2- activated Cl current in control conditions (filled squares, thin line) in the presence of 100 M ZnCl2 (crosses, dashed line) and in the presence of 100 M niflumic acid (open circles, thick line). Experimental protocol was as in A. Vertical bars indicate SEM. observed in the present study, but at holding potentials data on cGMP content in mesenteric arteries (Liu et of 60 mV the dominating current was an inward cur- al., 2002) with data on smooth-muscle cell water con- rent. Using a combination of ion substitution experi- tent (Aalkjaer and Mulvany, 1983) suggest that concen- ments and blockers it was possible to conclude that the trations in the range 2–4 M may be physiological. This current was a Cl current since the current reversal po- agrees well with the concentration range over which tential closely followed the Cl equilibrium potential, the present channel is regulated. Since the current was whereas extracellular removal of either Na or Ca2 elicited by four different means of increasing intracel- had no effect on the current. lular Ca2 it is unlikely that the role of cGMP is due to The pharmacological and biophysical characteristics an effect on the Ca2 release or Ca2 kinetics. In partic- of this Ca2-activated, cGMP-dependent Cl current ular since ionomycin could elicit the current in the were investigated and will be discussed in relation to presence of ryanodine it is unlikely that cGMP acts the previously described Ca2-activated Cl current in through modification of Ca2 release from the SR. smooth muscle (Wang et al., 1992; Large and Wang, Therefore, a more likely site of action of cGMP is on 1996; Greenwood and Large, 1999). the channel or a channel-related protein. Moreover, since current activation seemed to require phosphory- cGMP Dependence lation, was sensitive to PKG inhibitors, and could be The cGMP-dependent ICl(Ca) was evoked by calcium re- evoked by PKG, we believe that the effect of cGMP on lease from the SR in response to caffeine application, the current is mediated through PKG. through influx of Ca2 induced by ionomycin or BAY K There is no previous report on a cGMP-dependent

8644, and by direct activation with high-calcium pipette ICl(Ca) in smooth muscle. In cat tracheal smooth-muscle, solution. In every case the cGMP dependence was ap- cGMP inhibits a Ca2-activated Cl current, an effect parent and the EC50 value of 6 M (determined with not mediated by G kinase (Waniishi et al., 1998). That caffeine induced activation) is similar to that of cyclic current further differs from the present current in hav- nucleotide–gated channels elsewhere (Müller et al., ing a higher sensitivity to Cl-channel blockers and be- 1998, 2001), and substantially lower than that for ing insensitive to 500 M Zn2. Indirect evidence from cGMP modulation of calcium-activated potassium cur- rat aorta, based on the inﬂuence of the chloride gradi- rents (Schubert and Nelson, 2001). Recalculation of ent on contractile responses to noradrenaline in the

130 A cGMP-dependent Ca2-activated Cl Channel presence and absence of endothelium, also points to Cl F). The sequence Br I Cl F is typical an inhibitory role of cGMP on chloride currents (Lamb for a system with an I/Cl selectivity ratio close to 1, and Barna, 1998). In portal vein smooth-muscle cells, while the sequence I Br Cl F is characteris- both enhancement and inhibition of the volume-sensi- tic for systems with an I/Cl ratio higher than 1 tive chloride current by cGMP have been reported (Wright and Diamond, 1977). Our data had I/Cl ra- (Ellershaw et al., 2000). Cyclic AMP generally has been tios of 1.25 (permeability) and 1.41 (conductance), reported to activate chloride channels, especially the and the obtained selectivity sequences thus fit the theo- CFTR (Chen et al., 2001), but had no effect on the retical predictions well (Wright and Diamond, 1977). present current. Interestingly, the epithelial CFTR The data are consistent with selectivity being primarily channel was also shown to be activated by 100 M based on energy of hydration. cGMP via PKG (Lin et al., 1992). However, that channel Thiocyanate had a high permeability but a low con- had a different permeability sequence from the one ductance through the channel, indicating that it may studied here, and could also be activated by PKA. bind with high affinity within the channel. Thiocyanate In addition to the cGMP-dependent current, a shows a similar high affinity for the GABA receptor of cGMP-independent current was detected in the spinal neurons (Bormann et al., 1987). It also shows smooth-muscle cells from the rat mesenteric arteries, high affinity for the Xenopus oocyte calcium-activated and this was the only current detected in the smooth- chloride channel (Qu and Hartzell, 2000), although muscle cells from pulmonary arteries. This indicates both its relative permeability and conductance in the that not all vascular beds may have a prominent cGMP- oocyte channel are somewhat higher than in our data. dependent ICl(Ca) and also shows that the cGMP depen- The fact that both cGMP-dependent and cGMP-inde- dence was not a consequence of our experimental ap- pendent Cl currents were present in the same smooth- proach. The fact that the conventional ICl(Ca) was more muscle cells opens the possibility that the data for an- clearly detected with high calcium in the pipette than ion selectivity for the cGMP-dependent current might after caffeine application may indicate that the two cur- be contaminated by the cGMP-independent current. rents differ in their calcium sensitivity or are differently We never observed the conventional, cGMP-indepen- affected by calcium release. dent current in experiments using ionomycin to ele- 2 ICl(Ca) has been reported to be more stable in experi- vate [Ca ]i under control conditions, and therefore ments using the permeabilized patch technique than in used this protocol in the substitution experiments. It is conventional whole-cell experiments, where it tends to possible that a contaminating current may have be- show significant run-down (Janssen and Sims, 1992; come visible in the presence of a substitute ion. How- Large and Wang, 1996). This suggests the presence of a ever, none the substitutes used here give substantially soluble factor of importance for maintaining channel larger currents than chloride through the conven- activity. In our previous study (Peng et al., 2001) using tional, cGMP-independent current (Qu and Hartzell, permeabilized patches, the current studied here could 2000), with the possible exception of aspartate. Even be activated even after some time in the absence of 8Br- though we cannot entirely exclude a contribution of cGMP. In the present study using conventional whole- the conventional current to the permeability experi- cell experiments the current was not detectable 5 min ments, we therefore believe any such contribution to after breaking the membrane in the absence of cGMP. be small. This suggests that the permeabilized cells retained some endogenous cGMP, which was sufficient for par- Effect of Cl Channel Blockers tial activation of the current. However, we did not ob- There are no specific high-affinity blockers for Ca2- serve such a pronounced run-down in the pulmonary activated Cl channels available presently. Niflumic myocytes, where the current was totally insensitive to acid has been shown in several studies to be an effective cGMP. This makes it unlikely that the run-down of the inhibitor in micromolar concentrations (Pacaud et al., conventional ICl(Ca) can be explained by washout of 1989; Hogg et al., 1994; Lamb et al., 1994; Kirkup et al., cGMP. 1996). The cGMP-dependent ICl(Ca) was sensitive to both niflumic acid, DIDS, and IAA-94, but the potency Relative Anion Selectivity of these drugs was low. However, some Ca2-activated The relative permeability for all Ca2-activated chloride Cl channel clones are either insensitive or have a low channels depends on the ionic radius, and although sensitivity to niflumic acid (Cunningham et al., 1995). slightly variable the typical sequence is I Br This has been suggested to be due to a dual effect of ni- Cl F (Amédée et al., 1990; Arreola et al., 1996; flumic acid to simultaneously enhance and block ICl(Ca) Clapp et al., 1996; Frings et al., 2000). The permeabili- by binding to an external site, probably close to the ties obtained in our experiments under biionic condi- mouth of the chloride channel (Piper et al., 2002). tions, while not identical, were similar (Br I Whether this can explain the low sensitivity of the

131 Matchkov et al. cGMP-dependent ICl(Ca) to niflumic acid is unknown. superficial or points to structural similarities remains to The fact that not only niflumic acid, but also DIDS and be determined. IAA-94 exhibited a low potency could suggest that the structure of the channel may differ from other Ca2- Physiological Role activated Cl channels. The experiments in Figs. 6 and Since the chloride equilibrium potential of mesenteric 8 demonstrate clearly the difference in sensitivity to ni- artery smooth-muscle cells is about 30 mV (Videbaek flumic acid between the cGMP-dependent and -inde- et al., 1990; Chipperfield and Harper, 2000), opening pendent ICl(Ca). of these channels in a resting smooth-muscle with a membrane potential in the range of 50 to 60 mV Cation Sensitivity will cause depolarization, calcium influx, and contrac-

ICl(Ca) is known to be sensitive to block by several multi- tion (Pacaud et al., 1992; Wang et al., 1992; Lepretre et valent cations, such as gadolinium, lanthanum, cad- al., 1994; Kirkup et al., 1996). In the arteries studied mium, cobalt, and nickel applied to the extracellular here, calcium release is not essential for depolarization side of the membrane, whereas Zn2 has little effect in response to noradrenaline, and the transmembrane (Tokimasa and North, 1996; Waniishi et al., 1998). gradient for chloride has little inﬂuence on the mem- Lack of sensitivity to Zn2 was also found in our experi- brane potential at steady-state, whether at rest or dur- ments on pulmonary arterial myocytes (Fig. 8) and ing maximal activation (Nilsson et al., 1998). The for the cGMP-independent ICl(Ca) in mesenteric artery cGMP-dependent ICl(Ca) therefore is not likely to be im- smooth-muscle cells. In contrast, the cGMP-dependent portant for the tonic contractile response to receptor 2 ICl(Ca) had a high sensitivity to block by Zn and was activation. In contrast, the current has characteristics unaffected by cobalt. This pattern is known for voltage- that could suggest that it has importance for smooth- gated chloride channels (Kurz et al., 1997; Chen, muscle synchronization as we have suggested previously

1998), and suggests that the cGMP-dependent ICl(Ca) in (Peng et al., 2001). Synchronization is essential for co- 2 this respect is more like these chloride channels. ordinating oscillations in [Ca ]i in vasomotion, and we have suggested that this is brought about by changes in Voltage and Time Dependence membrane potential subsequent to released intracellular calcium activating the cGMP-dependent current The cGMP-dependent ICl(Ca) did not display the voltage (Peng et al., 2001). In this way this current may gener- dependence commonly seen for other ICl(Ca) (Ishikawa ate intermittent depolarizations maintaining synchrony and Cook, 1993; Arreola et al., 1996; Large and Wang, between the individual smooth-muscle cells in the ves- 1996; Greenwood et al., 2001). The voltage depen- sel wall. At the same time, by promoting continuous dence of ICl(Ca) depends on the calcium concentration variations in tone, formation of the latch state is pre- and at very high intracellular calcium concentrations vented, enabling the smooth-muscle to respond rapidly little rectification of ICl(Ca) is seen in parotid glands to, e.g., neural stimuli. Being regulated by two impor- (Arreola et al., 1996). However, in some experiments 2 tant pathways, [Ca ]i and NO/cGMP, this current is we fixed intracellular calcium at 900 nM where we potentially important in integrating endothelial and found no rectification, while the cGMP-insensitive cur- smooth-muscle control of vascular function. rent in the same experiments exhibited outward rectifi- It may seem surprising that cGMP promotes an in- cation (Fig. 6). Even though the voltage independence ward current leading to depolarization and vasocon- was unexpected, similar observations have previously striction rather than the vasodilatation normally associ- been reported for ICl(Ca) in hepatocytes (Koumi et al., ated with increased cGMP. However, it should be 1994) and in neuronal tissue (Krause and Welsh, 1990; pointed out that the net effect of elevating cGMP in Hallani et al., 1998). mesenteric small arteries is hyperpolarization and re- laxation. How these apparently opposing effects of Comparison with other Channels cGMP on membrane events are orchestrated under The cGMP-dependent ICl(Ca) is similar to the classical, physiological conditions is currently unknown. cGMP-independent ICl(Ca) in terms of being calcium de- In summary, the present study characterized a novel pendent, but distinct in terms of lack of voltage and Ca2-activated Cl channel with an obligatory require- time dependence, and in terms of its sensitivity to phar- ment for cGMP in its activation by intracellular cal- macological inhibitors. Its high sensitivity to inhibition cium. This channel has distinct properties reminiscent by divalent cations seems reminiscent of voltage-acti- of characteristics of both classical Ca2-activated and vated Cl channels. However, many of these character- voltage-gated Cl channels. The channel is characteristics are similar to what has been reported recently for ized by a high sensitivity to inhibition by Zn2 but a low members of the bestrophin family (Qu et al., 2003; sensitivity to inhibition by conventional chloride chan- Tsunenari et al., 2003). Whether this similarity is only nel blockers and exhibits no rectification, and is thus

132 A cGMP-dependent Ca2-activated Cl Channel different from the ICl(Ca) of pulmonary artery myocytes vated Cl channels-homing in on an elusive channel species. (Greenwood et al., 2001; this study). Further molecular Prog. Neurobiol. 60:247–289. characterization of the channel(s) responsible for this Greenwood, I.A., and W.A. Large. 1999. Modulation of the decay of Ca2-activated Cl currents in rabbit portal vein smooth muscle current could provide insight into structural elements cells by external anions. J. Physiol. 516:365–376. of importance for Cl channel function and for regula- Greenwood, I.A., J. Ledoux, and N. Leblanc. 2001. Differential reg- tion of vascular function in general. ulation of Ca2-activated Cl currents in rabbit arterial and portal vein smooth muscle cells by Ca2-calmodulin-dependent ki- This work was supported by the Danish Medical Research Coun- nase. J. Physiol. 534:395–408. cil (grant no. 9802388) and the Danish Heart Foundation (grant Gustafsson, H., M.J. Mulvany, and H. Nilsson. 1993. Rhythmic con- no. 98-1-2-12A-22593). The Water and Salt Research Center at tractions of isolated small arteries from rat: influence of the en- the University of Aarhus is established and supported by the Dan- dothelium. Acta Physiol. Scand. 148:153–163. ish National Research Foundation (Danmarks Grundforsknings- Gustafsson, H., and H. Nilsson. 1994. Rhythmic contractions in iso- fond). lated small arteries of rat: role of K channels and the Na,K- pump. Acta Physiol. Scand. 150:161–170. Lawrence G. Palmer served as editor. Hallani, M., J.W. Lynch, and P.H. Barry. 1998. Characterization of Submitted: 6 November 2003 calcium-activated chloride channels in patches excised from the Accepted: 29 December 2003 dendritic knob of mammalian olfactory receptor neurons. J. Membr. Biol. 161:163–171. Hogg, R.C., Q. Wang, and W.A. Large. 1994. Action of niflumic REFERENCES acid on evoked and spontaneous calcium-activated chloride and Aalkjaer, C., and M.J. Mulvany. 1983. Sodium metabolism in rat re- potassium currents in smooth muscle cells from rabbit portal sistance vessels. J. Physiol. 343:105–116. vein. Br. J. Pharmacol. 112:977–984. Amédée, T., W.A. Large, and Q. Wang. 1990. Characteristics of Ishikawa, T., and D.I. Cook. 1993. A Ca2-activated Cl current in chloride currents activated by noradrenaline in rabbit ear artery sheep parotid secretory cells. J. Membr. Biol. 135:261–271. cells. J. Physiol. 428:501–516. Janssen, L.J., and S.M. Sims. 1992. Acetylcholine activates non- Arreola, J., J.E. Melvin, and T. Begenisich. 1996. Activation of cal- selective cation and chloride conductances in canine and cium-dependent chloride channels in rat parotid acinar cells. J. guinea-pig tracheal myocytes. J. Physiol. 453:197–218. Gen. Physiol. 108:35–47. Kirkup, A.J., G. Edwards, M.E. Green, M. Miller, S.D. Walker, and Berridge, M.J., and A. Galione. 1988. Cytosolic calcium oscillators. A.H. Weston. 1996. Modulation of membrane currents and me- FASEB J. 2:3074–3082. chanical activity by niflumic acid in rat vascular smooth muscle. Bormann, J., O.P. Hamill, and B. Sakmann. 1987. Mechanism of an- Eur. J. Pharmacol. 317:165–174. ion permeation through channels gated by glycine and gamma- Koumi, S., R. Sato, and T. Aramaki. 1994. Characterization of the aminobutyric acid in mouse cultured spinal neurones. J. Physiol. calcium-activated chloride channel in isolated guinea-pig hepa- 385:243–286. tocytes. J. Gen. Physiol. 104:357–373. Britton, F.C., S. Ohya, B. Horowitz, and I.A. Greenwood. 2002. Krause, K.H., and M.J. Welsh. 1990. Voltage-dependent and Ca2- Comparison of the properties of CLCA1 generated currents and activated ion channels in human neutrophils. J. Clin. Invest. 85:

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134 A cGMP-dependent Ca2-activated Cl Channel